The present invention relates to a skin culture and a process for preparing the same. More particularly, the present invention relates to a skin culture matrix, a skin culture and processes for preparing the same for reconstructing or healing a defect tissue in an early stage by applying the matrix or the skin to a skin defect such as a burn, a wound, a decubitus or a skin ulcer.
Hitherto a medical treatment used in general as therapy for a skin defect reaching to an upper layer of dermis such as a superficial dermal burn is a cover of the skin defect with wound dressings, ointment gauze or the like. However, when there is caused a skin defect reaching to a lower layer of dermis such as a deep dermal burn, a dermal burn or a decubitus in at least the second grade, self-reconstruction in a cutaneous tissue by proliferation of epidermal cells can not be expected. Therefore, such a defect has been treated by debriding a slough or an abnormal granulation tissue, reconstructing a normal granulation tissue by covering the defect with an allogenic skin, wound dressings or the like, and then reconstructing a skin by performing autologous split-thickness skin graft. In case of performing the autologous split-thickness skin graft, however, because a skin is taken from a normal position of a patient, there arises a problem as to restoration of appearance that the scar remain at the position. Further, in case that a wound extends in a wide range, it is difficult to carry out the autologous split-thickness skin graft.
In order to solve the problem, there have been developed a technique wherein epidermal cells or fibroblasts are taken from a small skin piece and mass-cultured in a culture flask, and various skin cultures using these cultured cells.
The culture epidermis developed by Howard Green, James G. Rheinwald et al. is obtained by taking a skin of postage stamp size and mass-culturing cells derived from the skin in a culture flask to give an epidermal sheet. As to the sheet, the problem has been pointed out that taking ratio in autologous skin graft is low because basal cells being the most important cells are damaged by enzyme treatment in case of removing the sheet from the flask (see Yoshimitsu Kuroyanagi, Seitaizairyo (Biomaterial Feb. 9, 1991).
Also, Eugene Bell et al. (see Science, 211, pages 1052-1054, 1981) developed a skin culture by mass-culturing each of epidermal cells and fibroblasts in a culture flask which were obtained by separating them from a skin, and stratifying epidermal cells on a collagen gel into which fibroblasts has been incorporated to give a skin culture. However, there was a fault that a collagen gel extremely contracts during cell culture.
Further, Steven Boyce et al. (see SURGERY, 103, pages 421-431, April, 1988) developed a skin culture by mass-culturing epidermal cells obtained from a skin and stratifying the epidermal cells on a matrix in a form of sponge which was obtained by adding a small amount of chondroitin-6-sulfate to collagen.
Also, skin cultures having atelocollagen as a matrix are described in Japanese Unexamined Patent Publications No. 246371/1987 and No. 332561/1992. In Japanese Unexamined Patent Publication No. 246371/1987, a skin culture having an atelocollagen sheet as a matrix is described. In the above skin culture, however, there was a problem that taking ratio of epidermis is low in autologous skin graft. The reason is that the sheet has no penetrating pore, and nutrition being indispensable to cell proliferation and the like and being given from a covered wound face is hardly supplied through the sheet to epidermal cells on the opposite side of the sheet.
Also, in case of preparing a skin culture wherein only fibroblasts are taken into the skin, nutrition to be supplied from a covered wound face is hardly supplied throughout the skin culture. Therefore, proliferation and metabolism of fibroblasts are harmfully influenced and release of biologically active substances (e.g., growth factors, cytokines and the like) is not performed without hitch. These and the like often interfere with reconstruction of skin or good granulation.
There was the problem that a wound becomes a hotbed of infection by reason that exudate was excessively pooled in a wound and discharge of the exudate is not carried out properly, because of no penetrating pore.
In Japanese Unexamined Patent Publication No. 332561/1992, an atelocollagen sponge is described which is provided with a penetrating pore on purpose to solve the problem as described above. In case of providing the sponge with a penetrating pore, cells are flowed out through the pore and lost in being seeded. Therefore, pre-coating of the sponge with atelocollagen gel or the like before cell seeding is essential to the gel in order to compensate for such faults. There is the problem that such coating method is not only complicated but also expensive from the view points of cost for preparation of a culture skin. Therefore, development for more inexpensive and more simple alternative culture skin or preparation technique thereof has been desired.
The problems to be solved hitherto in a culture skin using collagen as a matrix were as follows:
1. contraction of a skin culture in cell culturing, PA1 2. the problem that nutrition supply throughout cultured cells from a covered wound face is not smooth, because a skin culture matrix becomes a barrier after covering a defect tissue of a skin with a skin culture, PA1 3. the problem that exudate is excessively pooled in a position where a defect tissue of a skin is covered with a skin culture after the covering, and PA1 4. the problem that cells flow out through a pore and are lost when being seeded, in the case a matrix is provided with a when penetrating pore in order to solve the above-mentioned points 2 and 3. PA1 a) a step in which a skin culture matrix comprising a collagen sponge, a collagen sheet or a collagen gel is prepared in a means having a projection to provide said matrix with a penetrating pore, PA1 b) a step in which a skin-derived cell is seeded and cultured on said matrix, and PA1 c) a step in which the skin culture is provided with a penetrating pore by the means having the projection before said skin culture is placed to cover a skin defect, if necessary, the steps a) and b) being simultaneously carried out. There is also provided a process for preparing a skin culture matrix comprising a collagen sponge, a collagen sheet or a collagen gel, in which said matrix is provided with a penetrating pore by a means having a projection before said matrix including a skin-derived cell is placed to cover a skin defect. PA1 a step in which an epidermal cell is seeded and cultured on one side of said matrix, or PA1 a step in which a skin-derived fibroblast is seeded and cultured on at least one side of side matrix and an epidermal cell is seeded and cultured on one side of said matrix; the step c) is accomplished by removing said projection from the matrix; the means having the projection is a support having a projection, a vessel having a projection, or a support or vessel having a projection with a removable sheet; the collagen is atelocollagen; and/or the culture skin matrix contains hyaluronic acid.
The present invention is made in order to solve the above points 1-4.
An object of the invention is to provide a skin culture for reconstructing or healing a defect tissue of a skin in an early stage by applying the skin culture to a skin defect such as a burn, a wound, a decubitus or a skin ulcer. More particularly, the object of the invention is to provide a skin culture which has a function by which there can be smoothly carried out nutrition supply from a covered wound face to cells of the skin culture and discharge of the exudate being excessively pooled in a wound, and is obtainable at a low cost without causing contraction.
A further object of the invention is to provide a skin culture matrix which can be used in the above-mentioned culture skin as a matrix.
A still further object of the invention is to provide processes for preparing the skin culture and the skin culture matrix.
These and other objects of the present invention will become apparent from the description hereinafter.